Magnetic CLEAs of β-Galactosidase from Aspergillus oryzae as a Potential Biocatalyst to Produce Tagatose from Lactose

Round 1

Reviewer 1 Report

This ms is concerned with the use of magnetic cross-linked enzyme aggregates (mCLEAs) of galactosidase, in particular in the conversion of galactose to tagatose. However, in the Introduction no mention is made of tagatose. In the Materials and Methods section, on p.16, the conversion of galactose to tagatose using a combination of galactosidase mCLEA and “immobilized L-arabinose isomerase” (mCLEA-LAI), i.e. magnetic CLEAs of LAI. The Materials and Methods section also contains extensive information regarding the preparation, structural characterization, storage, thermal and operational stability, etc.

In stark contrast, I cannot find any information in the manuscript regarding the preparation, characterization and properties of the mCLEAs of LAI. On p.13 it states: “The performance of ß-galactosidase mCLEAs from Aspergillus oryzae and L-arabinose isomerase from Enterococcus faecium [31] in the process of bioconversion of lactose to D-tagatose was studied in sequential bioreactors.” And on p.15: “For the immobilized L-arabinose isomerase, activity was determined according to Sousa et al (31)”

Apparently, details of the preparation and properties of L-arabinose isomerase mCLEA are given in ref. 31. which is from the same group. This should have been discussed in the Introduction, i.e. that they had previously prepared mCLEAs of the isomerase and now they are reporting the preparation of the galactosidase mCLEA and its combination with the LAI mCLEA and their use in the conversion of galactose to tagatose.

There is also a dearth of information on mCLEAs in general in the ms. There is an extensive literature on mCLEAs but one wouldn’t have guessed that from reading this paper. The authors refer to two reviews on CLEAs in general (refs 11 and 16 from 2016 and 2013, respectively) and one ref. from their own group on the synthesis of m-CLEAs (ref. 31). Ref. 12 from 2022 describes the synthesis of mCLEAs of laccase, cellulase, galactosidase and transglutaminase.

The literature on mCLEAs dates back to their invention which is described in a patent published in 2012 and the independent publication of Talekar, in Bioresour. Technol. 2012, 123, 542-547, in the sameFurther optimization of the technique was reported in a second patent, publihsed in 2018. Both of these patent refs can be found in a review published in Catalysts, 2019, 9, 261. The mCLEA technique has been widely exploited, e.g. in biomass conversion ( J. Solid State Chem. 2019, 270, 58-70) and many other applications. 

Indeed, the use of a combi-mCLEA of galactosidase and L-arabinose isomerase in the one-pot conversion of galactose to tagatose was reported in 2021 by Yadav and co-workers (Catal. Sci. Technol., 2021, 11, 2186-2194). The Yadav article contained a reference to the synthesis of mCLEAs of L-arabinose isomerase CLEA (ref. 31 in the submitted ms). However, the current authors make no reference to the Yadav paper in the submitted ms.

In short, this manuscript cannot  be accepted for publication until the authors have referred to all the relevant published literature on this subject and including a description of what is new and makes it worthy of publication in the light of the existing literature, in particular the Yadav paper.

 

Author Response

This ms is concerned with the use of magnetic cross-linked enzyme aggregates (mCLEAs) of galactosidase, in particular in the conversion of galactose to tagatose. However, in the Introduction no mention is made of tagatose.

Answer: We acknowledge the reviewer comment. Indeed, we have improved the introduction to add the information on tagatose production and importance.

 

In the Materials and Methods section, on p.16, the conversion of galactose to tagatose using a combination of galactosidase mCLEA and “immobilized L-arabinose isomerase” (mCLEA-LAI), i.e. magnetic CLEAs of LAI.

The Materials and Methods section also contains extensive information regarding the preparation, structural characterization, storage, thermal and operational stability, etc. In stark contrast, I cannot find any information in the manuscript regarding the preparation, characterization and properties of the mCLEAs of LAI.

Answer: We acknowledge the reviewer comment. We have updated the manuscript and added the suggested information.

 

 

On p.13 it states: “The performance of ß-galactosidase mCLEAs from Aspergillus oryzae and L-arabinose isomerase from Enterococcus faecium [31] in the process of bioconversion of lactose to D-tagatose was studied in sequential bioreactors.” And on p.15: “For the immobilized L-arabinose isomerase, activity was determined according to Sousa et al (31)” 

Apparently, details of the preparation and properties of L-arabinose isomerase mCLEA are given in ref. 31. which is from the same group. This should have been discussed in the Introduction, i.e. that they had previously prepared mCLEAs of the isomerase and now they are reporting the preparation of the galactosidase mCLEA and its combination with the LAI mCLEA and their use in the conversion of galactose to tagatose.

Answer: We acknowledge the reviewer comment. We have updated the manuscript and added the suggested information.

 

There is also a dearth of information on mCLEAs in general in the ms. There is an extensive literature on mCLEAs but one wouldn’t have guessed that from reading this paper. The authors refer to two reviews on CLEAs in general (refs 11 and 16 from 2016 and 2013, respectively) and one ref. from their own group on the synthesis of m-CLEAs (ref. 31). Ref. 12 from 2022 describes the synthesis of mCLEAs of laccase, cellulase, galactosidase and transglutaminase. 

The literature on mCLEAs dates back to their invention which is described in a patent published in 2012 and the independent publication of Talekar, in Bioresour. Technol. 2012, 123, 542-547, in the same Further optimization of the technique was reported in a second patent, publihsed in 2018. Both of these patent refs can be found in a review published in Catalysts, 2019, 9, 261. The mCLEA technique has been widely exploited, e.g. in biomass conversion ( J. Solid State Chem. 2019, 270, 58-70) and many other applications. 

Answer: We acknowledge the reviewer comment. The introduction was updated to add the information that the reviewer suggested.

 

Indeed, the use of a combi-mCLEA of galactosidase and L-arabinose isomerase in the one-pot conversion of galactose to tagatose was reported in 2021 by Yadav and co-workers (Catal. Sci. Technol., 2021, 11, 2186-2194). The Yadav article contained a reference to the synthesis of mCLEAs of L-arabinose isomerase CLEA (ref. 31 in the submitted ms). However, the current authors make no reference to the Yadav paper in the submitted ms.

In short, this manuscript cannot be accepted for publication until the authors have referred to all the relevant published literature on this subject and including a description of what is new and makes it worthy of publication in the light of the existing literature, in particular the Yadav paper.

Answer: We acknowledge the reviewer comment. We have updated the manuscript and added the suggested literature in discussion and introduction.

Reviewer 2 Report

·        According to the title of the publication, the immobilized enzyme is supposed to be used to obtain tagatose, but unfortunately there is no reference to tagtose in the paper's introduction.  Moreover, the introduction lacks information on the use of the magnetic CLEAs method to immobilize other enzymes.

·        Why glutaraldehyde and aldehyde dextran were chosen as a crosslinking agents?

·        The publication is too long in relation to the importance of the content presented, in my opinion, it should be halved. I suggest moving some things to SI. For example, Tables 2, 4, 6, 7 summarize Figures 1, 2, 4, 5 respectively. I therefore propose to move Figures 1, 2, 4, 5 to SI

·        How can you explain that the half-life for all immobilized enzymes is lower at 50C  (table 7) than at 60C (table 6)

·        In my opinion point 2.6 (Bioconversion of lactose into glucose and galactose ) is unnecessary, it adds nothing to the work

·        What was the method of separation of the heterogeneous catalyst between cycles (Figure 8)

·        The most interesting point about the synthesis of tagatose is described perfunctorily (2.10. Lactose bioconversion into tagatose by immobilized β-galactosidase and L-arabinose isomerase

Line 37-38: „The Aspergillus oryzae β-galactosidase has in the active site a glutamic acid residue that functions as an acid/base-catalyst and another one that functions as a nucleophile.” - does this mean that there are two glutamic acid molecules in the active centre or is there another amino acid? (if different, please give its name)

Line 37-38: “When within the active site, the β-galactose monomer makes hydrogen bonds with other residues around the domain [3,4].” I don’t understand this sentence.

Line 90; What is the precipitation rate and could it be expressed in percentages?

Author Response

According to the title of the publication, the immobilized enzyme is supposed to be used to obtain tagatose, but unfortunately there is no reference to tagtose in the paper's introduction.  Moreover, the introduction lacks information on the use of the magnetic CLEAs method to immobilize other enzymes.

Answer: We acknowledge the reviewer comment. We have updated the manuscript and added the suggested literature in discussion and introduction.

 

Why glutaraldehyde and aldehyde dextran were chosen as a crosslinking agents?

Answer: We acknowledge the reviewer comment. Further information about the use of those crosslinking agents were added to introduction.

 

The publication is too long in relation to the importance of the content presented, in my opinion, it should be halved. I suggest moving some things to SI. For example, Tables 2, 4, 6, 7 summarize Figures 1, 2, 4, 5 respectively. I therefore propose to move Figures 1, 2, 4, 5 to SI

Answer: We acknowledge the reviewer comment. The manuscript was modified, and the figures are now provided as SI.

 

How can you explain that the half-life for all immobilized enzymes is lower at 50C  (table 7) than at 60C (table 6)

Answer: We acknowledge the reviewer comment. Actually the half-lives at 50C are higher than at 60C. The unit in table 7 was different and this may be confusing. We have changed to minutes (and not hours).

 

In my opinion point 2.6 (Bioconversion of lactose into glucose and galactose) is unnecessary, it adds nothing to the work

Answer: We acknowledge the reviewer comment, however, we would like to maintain the results in the manuscript. It is important to see if the rate of lactose hydrolysis is compromised by mass transfer limitations when using the heterogeneous biocatalysts. Although we did not conduct a series of experiments to confirm diffusion limitation, the initial rate of  lactose hydrolysis may give us a clue. This discussion is now added to the manuscript.

 

What was the method of separation of the heterogeneous catalyst between cycles (Figure 8)

Answer: We acknowledge the reviewer comment. This information was added to the manuscript on material and methods, when describing operational stability.

 

The most interesting point about the synthesis of tagatose is described perfunctorily (2.10. Lactose bioconversion into tagatose by immobilized β-galactosidase and L-arabinose isomerase

Answer: We acknowledge the reviewer comment. We have improved discussion on this topic.

 

Line 37-38: „The Aspergillus oryzae β-galactosidase has in the active site a glutamic acid residue that functions as an acid/base-catalyst and another one that functions as a nucleophile.” - does this mean that there are two glutamic acid molecules in the active centre or is there another amino acid? (if different, please give its name)

Answer: We acknowledge the reviewer comment. The information was added to the manuscript, there are 2 Glu residues, Glu200 and Glu298.

 

Line 37-38: “When within the active site, the β-galactose monomer makes hydrogen bonds with other residues around the domain [3,4].” I don’t understand this sentence.

Answer: We acknowledge the reviewer comment. The idea was to describe the interaction of the substrate molecule with the residues in the active site. The sentence was modified for clarity.

Line 90; What is the precipitation rate and could it be expressed in percentages?

Answer: We acknowledge the reviewer comment. Indeed, what we meant was that immobilization yield was 100 %. That is now written in the manuscript.

Round 2

Reviewer 1 Report

The authors have now included the Yadav reference but what the authors fail to do is mention that the Yadav paper describes the use of m-combiCLEAs of the galactosidase and the arabinose isomerase to produce tagatose directly from lactose. And it would be useful if they could briefly say what their manuscript adds to the existing literature considering the paper of Yadav.

Author Response

Thanks for your important and constructive criticisms that allowed us to greatly improve the scientific quality of our work.

Reviewer #1:

The authors have now included the Yadav reference but what the authors fail to do is mention that the Yadav paper describes the use of m-combiCLEAs of the galactosidase and the arabinose isomerase to produce tagatose directly from lactose. And it would be useful if they could briefly say what their manuscript adds to the existing literature considering the paper of Yadav.

Answer: We acknowledge the reviewer comment. We have improved the introduction to add information on tagatose production and its importance. The following text was added:

Rai et al. [11] describe, for the first time, a magnetic CLEA that combines the enzymes L-AI and β-Gal and that can be applied directly in the transformation of lactose into D-tagatose. The authors, however, used glutaraldehyde as a crosslinking agent for the preparation of Combi-mCLEA, which may be a disadvantage since glutaraldehyde is reported as a toxic/hazardous substance [25].

Although dextran-aldehyde has been used to prepare CLEAs[40], the crosslinking step can be a bottleneck since it involves the reaction of reactive primary amino groups on the protein surface, which may not be available in large amounts. Thus, in such cases, the preparation of CLEAs is not simple and depends on the enzyme in question. To the best of our knowledge, the use of dextran-aldehyde as a non-toxic alternative for glutaral-dehyde for the preparation of mCLEAs of β-galactosidase has not been reported.

Reviewer 2 Report

it is ok

Author Response

Thanks for your important and constructive criticisms that allowed us to greatly improve the scientific quality of our work.